Note: Descriptions are shown in the official language in which they were submitted.
8~3~
FIELD OF THE INVENTION
This invention relates to a process for the prepa-
ration of an antitumor agent from shellfish or mollusc o-n
a large scale and to the antitumor agent thus prepared.
BACKGROUND OF THE INVENTION
Most o~ known antitumor agents are oY low molecular
weights and rely on their direct cytotoxic effect on tumor
cells in vivo~ so that they have a high toxicity against
host animals, giving unfavorable effect thereon. On the
other hand, some polysaccharides having a relatively high
molecular weight, a low toxicity and significant immuno-
potenciating and antitumor properties have been used in
the form of a partially purified powder derived ~rom diverse
sources including higher plants, fungi, lichen, bacteria
and yeasts by extraction, but they are narrow in antitumor
spectra and thus have limited applications in practical
purposes.
It has already been reported that some antitumor
materials are obtained from shellfish (see Japanese Patent
Publication No. 8088/82 and Japanese Patent KOKAI Nos.
41314/79 and 41315/79). According to these publications,
the antitumor materials are prepared by a process comprising
(1) removing shells from raw shellfish with or without
heating the latter, (2) removing the liver from the meat,
(3) finely cutting or grinding the resulting meat in an
aqueous solvent such as water and a saline solution at a
-- 1 --
~38~
low temperature by means of a blender and the like,
optionally I ollowed by sonication or other physical impact
treatment whereby to attain high homogenization of the
finely divided meat pieces wi-th good extraction of desired
antitumor materials in the cold aqueous solvent, (4) remov-
ing all the water-insoluble materials including the exhausted
meat pieces from the resulting mixture by centrifuging or
other means and (5) isolating the desired antitumor mate-
rials from the ex-tract thus obtained by purification means
known for the ~solation of protein substances such as
dialysis, ultrafiltration, gel-filtration and column
chromatography. The antitumor materials thus obtained
from shellfish are interested in that they have broad anti-
tumor spectra and high therapeutic activities. However~
the processes hitherto proposed for the preparation of
these materials as above-mentioned have such disadvanta~es
that they have to spend the edible portions of shellfish
being expensive, require complicated procedures for the
extraction of desired materials and involve problems on
disposal of the spent residue of meat pieces in large
amount, so that it seems di~ficult to provide these mate-
rials on an industrial scale through these processes.
We have made our investigations with the intention
of providing a process acceptable for the purpose of
industrial production of antitumor materials of a kind
similar to those derived from the meat of shellfish as
3LZ13885
above-men-tioned and h~ve now found that the llquid portion
which comes from coo~ing of raw shellfish carried out in
a hot aqueous solvent or with vapor of such solvent to take
up edible portion thereof and which is to be discarded as
waste can serve as raw material from which water-soluble,
macromolecular glycoprotein subs-tances useful as antitumor
agent is recovered and that these substances have a range
of molecular weights within the limits of from 10,000 to
300,000 and can be recovered efficiently by certain combi-
nation of purification techniques. The liquid portioncoming from cooking of raw shellfish has hiterto been
discarded as waste with costly additional treatment
required to avoid environmental contamination and is
hereinafter referred to as "waste liquor".
SUMMARY OF THE INVENTION
Accordingly, it is the main object of this invention
to provide a process for the preparation of an antitumor
agent from waste liquor, i.e. the liquid portion coming
from cooking of raw shellfish. Another object of this
invention is to provide an antitumor agent consisting
essentially of water-soluble 7 macromolecular glycoprotein
substances having a range of molecular weights within the
limits of from lO,OOO to 300,000. These and other objects
of this invention will become clear from the following
descriptions.
According to the general aspect of this invention,
388~
therefore~ there is provided a process for the preparation
of an antitumor agent consisting essentially o~ water-
soluble, macromolecular glycopro-tein substances having a
range of molecular weights within the limits of from
10,000 to 300,000 ~rom shellfish which comprises recovering
the liquid portion which comes from cooling o~ raw shell-
fish in a hot aqueous solvent or with vapor of such solvent
and which is to be discarded as waste, concentrating the
liquid portion thus recovered to a smaller ~olume or to
dryness to yield a concentrate or dry powder and isolating
the water-soluble, macromolecular glycoprotein substances
from the concentrate or dry powder.
BRI~F DESCRIPTION OF THæ DRAWINGS
~_ . . .
Figures 1 and 2 show two examples of elution pattern
depicted on elution with v~rious concentrations of aqueous
sodium chloride solution o~ substances adsorbed on a basic
anion-exchanger in ion-exchange chromatQgraphy of an aque-
ous solution of dry powder derived from waste liquor of
cooking of raw scallop, wherein the abscissa axis repre-
sents fraction numbers of the eluate, the ordinate axis
represents absorbance shown as optical density, the white
circles show absorbance at wave length of 490 nm of each
eluate fraction colored by the method of Dubois et al.
(see J.Anal. Chem., 28, 350~356 (1956)) as an indicator
of the carbohydrate conten-t of fractions and the black
circles show absorbance at wave length of 750 nm of each
~3~
eluate fraction colored by Lowry method as an indlcator
of the protein content of fractions.
Figure 3 shows an elution pa-ttern depicted on
elution with phosphate buffer solutions in gel-filtration
of the fraction C of Fig. 1 using a gel having a fractio-
nation range of molecular weights of 1,500~100,000, where-
in abscissa and ordinate axes and the white and black
circles represent same meanings as those given in the
preceding figures.
Figures 4 and 5 show ultraviolet absorption spectrum
and infrared absorption spectrum, respectively, of water-
soluble glycoprotein substances having a range of molecular
weights of 10,000~300,000 which were obtained by gel-
filtration of the fraction C in Fig 1 followed by another
gel-filtration to remove fractions having molecular weights
higher than 300,0000
DETAILED DESCRIPTION OF THE INVENTION
The liquid portion coming from cooking of raw
shellfish to be used as starting ma-terial according to the
process of this inven-tion may include those by-produced
when fresh or raw shellfish which may have been heated,
i~ desired, is cooked or heat-treated in a hot aqueous
solvent or with vapor of such solvent for the purpose of
obtaining edible portions thereof. The hot aqueous solvent
to be used as heating medium may include hot or boiling
water, steam and other hot aqueous solvents and vapor of
L3~
such solvents,
The term "shellfish" used herein includes not only
those so-called in a narrow sense, i.e. those having outer
shell ~rom among Phylum Mollusca, but also those so-called
in broad sense, i~e. any of Mollusca including those living
in sea and fresh water, Examples of shellfish are an
abaline (Haliotis discus)) a horned turban (Batillus
cornutus), a mud-snail ~SIL~A o~^l~dl ~ chinensis malleata),
_ _
Semisulcospira libertina, a hepatic moon shell (Neverita
(Glossaulax) didYma), an ivory shell (~ p~ ,ja~onica)~
__
a ribbed ark shell ~a~ broughtonii), a Japanese
scallop (Pecten albicans), a scallop (Patinopecten yessoen-
sis), a pearl oyster (Pinctada fucata~, a hard shell mussel
(My~a~ coruscus), an Asian hard clam (Meretrix lusoria),5 a Japanese little neck (~ a~ ), a surf clam
chinensis), a silver mouthed turban shell
(Marmarostoma ~ )3 Turbo petholatus reevei, and
Meretrix lamarcki.
The step for preparing the starting material to
be used according to this invention, i.e. -the liquid por-
tion which comes from cooking of raw shell~ish and which
is to be discarded as waste~corresponds to the step for
taking up edible portion of shellfish by cooking or heat-
treating raw shellfish in a hot aqueous solvent or with
vapor of such solvent and may -therefore be carried out by
any method kno~m or conventionally used in the art.
~2~3~
Of course, it is important and pre~erred ~or the purpose
of this invention to control the cooking step so as to
yield the desired water-soluble, macromolecular glyco-
protein substances having a range of molecular weights
within the limits of from 10,000 to 300,000 (hereinafter
referred to as "antitumor substances") in as much amount
as possible without impairing the primary object ~or the
coo~ing step. Thus, the following explanation on details
of the cooking step will be direc-ted to preferred embodi-
ments for attaining the purose of this invention.
In order to take up edible portion of shellfish and
to recover the liquid portion containing antitumor sub-
stances by cooking or heat-treating (hereinafter referred
to as "cooking") raw shellfish in a hot aqueous solvent
or vapor of such solvent~ the cooking operation is effected
in one or more steps, in each of which the shellfish is
brought into con-tact with a hot aqueous solvent in the
form of liquid and/or vapor which serves as both heating
medium and extracting solvent. The method of contact
between shellfish and hot aqueous solvent may be selected
as desired~ for example from among those of direct exposure
to solvent vapor, direct pouring of hot solvent and
immersion in-to hot solvent. The hot aqueous solvent may
be used in the form of either liquid or vapor or both.
For the purpose of this invention, the raw shellfish
may be used in its entirety, i.e. in the shell; or a
~2~3~
shelled form with or without liver, as desired. According
to this invention, therefore, all the edible portions, i.e.
meat, ligament and the like, of shell:~ish can be used for
food after the cooking operation because no cutting or
grindin~ of shellfish is reauired for cooking.
Usually, the cooking ~ay be carried out at a tem-
perature of about 50-120C, preferably about 60~120C.
As above-~entioned, the cooking operation may be
carried out in one or more, e.g. two or three, steps,
the -cwo steps being preferred. In a one-step process,
raw shellfish may be treated as desired by subjecting it
to e~posure to vapor of an aqueous solvent, by pouring a
hot aqueous solvent thereon or by immersion -thereof into
a hot aqueous solvent, whereby to take up all the edible
portions of shellfish and to recover the liquid portion
from the treated mixture. Typical cooking condi-tions
preferably used for one-step process are given in Table 1.
In a two-step process, the first step may follow the one-
step process and the second step may preferably be carried
out by immersion under heating of the shellfish derived
from the ~irst step in a hot aqueous solvent preferably
containing sodium chloride whereby to take up all the
edible portions of shellfish and to recover the first and
second liauid portions from the first and second coo~ing
s'ceps, respectively. Typical cooking conditions preferably
used for two-step process are given in Table 2. It 1~till
be most preferred for the two-step process to carry out
the first step by exposure of raw shellfish to vapor of
an aqueous solvent and the second step by immersion of
the shellfish thus treated and taken up into a hot aqueous
solvent containing sodium chloride, e.g. in a concentration
of 0.2~20% under heating. Such a t~ro-step process is
advantageous in that the amount of aqueous solvent to be
used is minimized with a high efficiency of extraction of
antitumor substances, that -the step for concentration of
the resulting liquid portion required is simplified and
shortened, that the heat e~ficiency is high and that the
apparatus or e~uipment to be used is less complex.
3~
Table 1 T~pical cooking conditions for one-step ~rocess
Embodiment 1 ¦ Embodiment 2 Embodiment 3
_ _. ..._ . .
Starting material Raw shellfish Raw shellfish Raw shellfish
solvent Hot vapor Hot liquid Hot liauid
Method of contact Directly ex- Pour hot liq- Heat starting
between starting pose starting uid to start- material in
material and material to ing material hot liquid
aqueous solvent hot vapor
Temperature of
aqueous solvent About 100~130l About 50~100 About 50~130
used C
(Most preferred (About 105~ 1 (About 90~ (About 90~
temperature) 120) l~ 100) 100)
Ratio of aqueous About 0.03~ 1 About 0.05~ About 0.6~
solYent/starting o . 8 ! lo 20
material, wt/wt
(Most preferred ~About 0.05~ 1 (About 0.07~ (About 1~15)
ratio) 0.5) ' 1.0)
Time of contact
between start- About 3~120 i About 3~120 About 3~120
ing material and
aqueous solvent,
minute
(Most preferred (About 5~60) ¦ (About 5~60~ (About 5~60)
time)
Cooking tempera- About 50~120 About 50~100 About 50~120
ture, C
(Most preferred (About 60~ (About 60~ (About 60~
tO~er~tD~I l2~) _ _ _ _
-- 10 --
31~
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o ~ rl 0 ~ ~ h 0
0~ h ~ ~ h O h^
h aJ h ~ h ~ h ~ h a~
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-- 12 --
~%~3~
Cooking pressure may be atmospheric, subatmospheric
or superatmospheric as desired.
The aqueous solvent to be used for the cooking may
include water, saline solution, sea water, aqueous solutions
of various water-soluble salts (preferably in a concen-
tration of about 0.5~20% by weight), buffer solutions and
aqueous solutions of water-soluble solvents, typically a
lower alcohol such as ethanol (preferably in a concen-
tration of about 1~50% by weight), the preferred aqueous
solvent being water or saline solution.
The liquid portion coming ~rom the cooking of raw
shellfish as above-mentioned, including both the first and
second liquid portions in cases of -two-step cooking process,
which contains antitumor substances may be concentrated
to a smaller volume or to dryness to yield a concentrate
or dry powder. For this purpose, the liquid portion may
be heated at a temperature of about 30~100C under appro-
priate pressures to concentrate it~ treated by dialysis
or ultra~iltration to remove salts 7 or dried by hot-air
drying such as spray-drying~ or freeze-drying to give dry
powder. In cases where the liquid portion is recovered
in the form of dry powder in a hot-air dryer, the hot air
inlet temperature is usually about 200-350C, preferably
about 250~310C and the outlet temperature thereof is
usually about 80~170C, preferably about 100~150C and
the contact (residence) time is usually about 5~80 seconds,
s
preferably about 5-50 seconds. Under these conditions,
the antitumor substances to be recovered are stable with-
out decomposition.
According to this invention, the concentrate or
dry powder thus derived from the liquid portion which
comes from the cooking of raw shellfish as waste liquor
is treated to isolate antitumor substances which are
water-soluble, macromolecular glycoprotein substances
having a range of molecular weights within the limits of
from 10,000 to 300,000. This may be done by an appropriate
combination of various known purification techniques, but
we have found a certain specific combination of purification
processes which is capable of isolating the desired anti-
tumor substances from the concentrate or dry powder most
efficiently and economically.
According to a preferred aspect of this invention,
therefore, the treatment for isolating the antitumor sub-
stances comprises dissolving the concentrate or dry
powder in an aqueous solvent such as water or a bu~fer
solution and subjecting the resulting aqueous solution to
a series o~ chromatographic treatments comprising an ion-
exchange chromatography with a basic anion-exchanger and
a gel-filtration to collect fractions which are adsorbed
on the basic anion-exchanger and which have a range of
molecular weights within the limits of from 10,000 to
300,000~
L3~5
The sequence of the ion-exchange chromatography
and gel-filtration may be chosen as desired) but the ion-
exchange chromatography followed by gel-filtration is
particularly preferred.
Prior to the ion-exchange chromatography or gel-
filtration, the aqueous solution of concentrate or dry
powder should preferably be pretreated in order to remove
insoluble parts and inorganic salts contained therein for
assuring a smooth operation of the subsequent treatments.
Thus, according to the most preferred aspect o~ this
invention, the treatment for isolating the antitumor sub-
stances comprises dissolving the concentrate or dry powder
in an aqueous solvent such as water or a phosphate buffer
solution, purifying the resulting aqueous solution from
insoluble materials and inorganic salts contained therein,
subjecting the resulting aqueous solution to an ion-
exchange chromatography with a basic anion-exchanger to
adsorb water-soluble, macromolecular glycoprotein substances
on the ion-exchanger followed by elution of the substances
and subjecting the eluted substances to a gel-fil~ration
to collect fractions having a range of molecular weights
within the limits of from 10,000 to 300,000.
The water-soluble glycoprotein substances adsorbed
on basic anion-exchanger in an ion-exchange chromatography
may be eluted desirably with an aqueous sodium chloride
solution having a concentration of about 0.07-0.4 M,
- 15 -
~2~3~
preferably about 0.1~0~38 M and most preferably about
001~0.3 M as eluent.
Details of treatment for isolating the desired
antitumor substances from an aqueous solution of the
concentrate or dry powder will be given below, in which
the evaluation of antitumor activity of the antitumor
substances isolated was made by the following method
unless otherwise stated~
Thus~ ICR mice (female, 6 weeks aged) are inoculated
intraperitoneally with Sarcoma 180 tumor cells, followed
by withdrawing the reproduced tumor cells together with
ascites from the inoculated mice 1 week after the tumor
cell inoculation.
0.05 Millilitres (about 4 x 106 cells) of the 7-day-
old Sarcoma 180 ascites tumors so cultured and collectedare subcutaneously transplanted into the right groin of
female ICR mice, weighing about 2~ g. The test samples
dissolved in a physiological saline in adequate concen-
trations (injection volume, 0.1 m~) are injected into the
tumor site on days 5, 7, and 9 after tumor inoculation,
by which time the tumors have reached an average diameter
of 5 mm. At the end of week 5, the mice are killed and
the tumors dissected out and weighed. The inhibition
ratios are calculated by use of the following formula:
Inhibition ratio (%) = ~A - B)/A x 100,
- 16
~Z~l3~
where A is the average tumor weight of the control group
and B is that of the treated group. Complete regression
indicates the ratio of the number of mice shoT~ing complete
tumor regression to the total number of mice tested.
The removal of insoluble materials from the aqueous
solution of the concentrate or dry powder may be carried
out by any known technique such as centrifuging, ~iltration
and decantation. The removal of inorganic salts from the
aqueous solution may also be effected by any known technique
such as gel-filtration using a gel capable of fractionat-
ing low-molecular weight compounds such as inorganic salts
and dialysis through a cellulose membrane against distilled
water, In one embodiment useful for the removal of in-
soluble materials and inorganic salts, the concentrate or
dry powder of the liquid portion coming from the
cooking of raw shellfish is dissolved in an aqueous solvent
such as water or a phosphate buffer solution, preferably
a 0~01-0.1 M, more preferably 0.01~0.05 M phosphate buffer
solution containing 0~0.15 mo~/~ of sodium chloride and
having a pH of 7.0~7.5 so as to gi~e a concentration of
about 10~25% by weight of the concentrate or dry powder
on the dry weight basis. A phosphate buffer solution is
an aqueous solution of a phosphate mixture of potasslum
dihydrogenphosphate and disodium hydrogenphosphate. The
resulting solution is centrifuged to remove insoluble
materials as precipitate and then chromatographed by
- 17 -
1518~
passing through a column packed with a gel for ~el-
filtration capable o~ fractionating substances having
molecular weights in the range of about 500~5,000, e.g~
Sephadex G-25 (Sephadex is a registered trade mark of
Pharmacia ~ine Chemicals Co., Uppsala, Sweden) which is
a gel obtained by three-dimensionally crosslinking dextran
with epichlorohydrin, followed by eluting with the same
buffer solution as above so as to collect fractions eluted
before the rise in the electric conductivity of the eluate
is observed. Thus, the separation of fractions of in-
organic salts from those of organic substances may be
ef~iciently achieved. ~ny other measure is applicable
in place of the measure of electric conductivity to deter-
mine the boundary for the said separation.
The fractions containing organic substances o~
relati~ely high molecular weights thus obtained are then
chromatographed by ion-exchange chromatography by passing
through a column packed with a basic anion-exchanging gel
having a high upper limit of molecular weights to be
fractionated, e~g. about 500 7 000~ 1 ~ 000 ~ 000 9 such as those
having diethylaminoethyl or aminoethyl group as an ioniz-
ing group. A ~ypical example of such anion-exchanging gel
is DEAE-Sepharose CL~6B (Sepharose is a registered trade
mark o~ Pharmacia Fine Chemicals Co.) which is a gel
obtained by three-dimensionally crosslinking agarose with
2,3-dibromopropanol followed by introducing diethylaminoethyl
~L3i!3~
group through an ether linkage and which has chloride ion
as the counter ion, the upper limit of molecular weights
to be fractionated of about 1 x 106 and the total exchange
capacity of 15 + 2 meq/100 m~. Non-adsorbable substances
which are mainly composed of polysaccharides and low ion-
attractive substances which are eluted with the salt con-
centration of eluent used are passed through the column
without being adsorbed thereon and are therefore removed
~rom adsorbable substances. Then, the substances adsorbed
on the gel are eluted with an eluent which is a buffer
solution containing sodium chloride, for example a 0.01~
0.1 M phosphate buffer solution containing 0.07-0.4 mo~
preferably 0.1~0.38 mol/~ and most preferably 0.1~0~3 mo~/~
of sodium chloride and having a pH of 7.0~7.5. The frac-
tionation of the eluate at respective ion concen-trations
is monitored by ultraviolet ray detector and the like.
The NaC~ concentrations in the buffer solution
used to elu-te the desired substances in the ion-exchange
chromatography above~mentioned are determined on the
basis of the result of the following experiment.
The fractions containing organic substances of
relati~ely high molecular weights which were obtained by
centrifuging followed by gel-filtration with Sephadex
G-25 of the liquid portion which came from the cooking of
scallops ~ ) were freeze-dried.
The resulting dry powder (5 g) was dissolved in a 0.01 M
-- 19 --
~Z~8~
phosphate buffer solution (20 m~) having a pH of 7.0,
and the resulting solution was passed through a column
of DEAE-Sepharose CL-6B equilibrated with a 0.01 M phos-
phate buffer solution. Chroma-tographic elution was effected
by gradient elution with eluents containing sodium chloride
in continuously varying concentrations, giving an elution
pattern shown in Fig. 1. Thus, four fractions, A, B, C
and D, which were eluted at sodium chloride concentrations
of 0, 0~0.1, 0.1~0.25 and 0.25~0.38 mo~/~, respectively,
were collected.
The fractions A, B, C and D so obtained were freeze-
dried to form four powders D-A, D-B, D-C and D-D, respec-
tively, and then evaluated for their antitumor activities
according to the bioassay method hereinbefore stated in
respect of Sarcoma 180 solid tumor. The test results
obtained are tabulated in Table 3 below~
- 20 -
3~2~
Table ~
.... , _ , ...................... . .. _
Test Dose Average Tumor Complete
Sample (mg/mouse x tumor Inhibi- regres-
times ) weight (g) ratio (%) sion
... . , ., .. _ ......
D-A 10 x 4 6.68 12.5 0~4 .
D-B 10 x 4 6.22 18.5 o/3
D-C 15 x 22 0.88 88.5 2/4
D-D 15 x 22 2090 62.0 1/4
. . _. ,.. _ __~ __.
Control . _ _ _ 7~63 ........... _ 0/4
(Note)* times: 5, 7, 9 and 11 days after the tumor trans-
plantation.
The above results indicate that the freeze-dried
powder (D-C) obtained from fraction C eluted from DEAE-
Sepharose column with OoOl M phosphate buffer solutions
containing 0.1 M - 0.25 M sodium chloride has a strong
antitumor activity among the samples (D~A), (D-B) and
(D-D), as it gave a 88.5~ tumor inhibition ratio with
complete regression in 2 of 4 mice tested.
Further, the same fractions as used in the preceding
ion-exchange chromatographic operation~ i.e. the fractions
containing organic substances which were obtained by
centrifuging followed by gel-filtration o~ the liquid
portion which came from the cooling of scallops were
21 -
~2~
dissolved in a volume of a 0.01 M phosphate buf~er solution
(pH 7.5), followed by chromatography in a column of DEAE-
Sepharose CL-6B developed with 0.01 M phosphate buffer
solutions (pH 7.5) containing different concentrations of
sodium chloride, with the sodium chloride concentration
being stepwise changed from 0.07 M to 0.25 M to 0.3~ ~,
and with the eluate being collected in 15 m~-fractions.
The elution pattern obtained in this chromatography is
shown in Figure 2. Further, the fractions eluted with
0.01 M phosphate buffer solution containing 0~25 M sodium
chloride were sub-divided into four fractions, so that
the fractions Nos, 130~152 where no absorption peak was
observed were combined together as fraction A; the fractions
Nos. 153-162 where the absorption peak of carbohydrates
was observed were combined together as fraction B; the
fractions Nos. 163-176 where the absorption peak of pro-
teins was obser~ed were combined together as fraction C;
and the last fractions Nosl 177~210 were combined together
as fraction D. These fractions A, B, C and D were freeze-
dried to form four powders DS-A, DS-B, DS-C and DS-D9
respectively, and then evaluated for their antitumor
activities against Sarcoma 180 solid tumor according to
the bioassay ~ethod stated hereinbefore~ The test results
obtained are shown in Table ~ below.
~388~i
Ta
TestDose Average Tumor Complete
Sample(mg/mouse tumor Inhibi- regres-
x times) weight (g) tion sion
_ ~ _ ratio (%)
DS-A 10 x 3 2.79 72,8 0/6
DS-B 10 x 3 2.95 71.2 0/6
DS-C 10 x 3 2.07 79.8 1/3
DS D 10 x 3 6.66 35.1 0/5
.... ~ .. ___ . . _
Control _ 10.26 _ 0/7
~. . . .___ . . . _ .. _
From the above results~ it is seen that fraction
DS-D showed a weak antitumor activity, while fractions
DS-A, DS-B and DS-C showed marked antitumor activities.
The above experiments indicate that when the DEAE-
Sepharose column was eluted with a phosphate buffer solu-
tion (pH 7.5) containing about 0.07~0.4 M sodium chloride,
the desired antitumor-active substances were concentrated
effectively.
The aqueous solution containing the desired sub-
stances thus obtained by the ion-exchange chromatography
is then subjected to gel-filtration to isolate those sub-
stances having a range of molecular weights within the
limits of from 10,000 to 300,000. For this purpose, the
aqueous solution is previously treated for the removal of
inorganic salts and for the concentration to a smaller
- 23 -
3~
volume or to dryness. The removal of inorganic salts or
desalting may be carried out in the similar manner as
above, for example by gel-filtra-tion or dialysis.
One typical embodiment of the pretrea-tment at this
stage comprises concentrating the aqueous solution at a
temperature below 40C9 preferably below 20C, under a
reduced pressure to about 1/5 - 1/6 volume, passing the
concentrated solution through a column of gel for gel-
filtration capable of fractionating substances having
molecular weights of about 500~59000, e.g. Sephadex G-25,
eluting the column with distilied or deionized water to
separate fractions con-taining inorganic salts from those
containing desired organic substances by monitoring the
electric conductivity of eluate fractions, concentrating
the fractions containing desired organic substances at a
temperature below 40C, preferably below 20C under a
reduced pressure to about 1/5 ~ 1/6 volume and freeze-
drying the concentrate to form dry powder. The yield of
the dry powder is about 1/60 ~ 1/100 based on the con-
centrate or the dry powder obtained from the liquidportion which comes from the cooking of raw shellfish.
The subsequent gel-filtration for the isolation of
desired organic substances having a desired range of
molecular weights is preferably carried out by using a gel
capable of fractiona-ti~g substances having molecular
weights of 1,500~1009000, e.g. Sephadex G-75 (Sephadex
- 24 -
3~38S
is a registered trade mark of Pharmacia Fine Chemicals Co.)
which is a gel obtained by three-dimensionally crosslinking
dextran with epichlorohydrin. According to one typical
embodiment of this gel~~iltration step, l g of the dry
powder which is obtained by desalting and freeze-drying
fraction D-C derived from elution in the ion~exchange
chromatography is dissolved in 5 m~ of a 0~0.1 M phosphate
buffer solution having a pH of 7.0~7,5. The resulting
solution is passed through a column of Sephadex G-75 which
has been equilibrated with a 0~0.1 M phosphate bu~fer
solution having a pH o~ 7.0~7.5. The column is then eluted
with a 0-0.1 M phosphate buffer solu-tion having a pH of
7.0-7.5 as eluent. One example of elution patterns given
in such gel-filtration is shown in Fig. ~. Thus, three
fractions A, B and C were collected9 the initial fraction
A showing small peaks of protein and carbohydrates, the
middle frac-tion B showing little or no peak of carbohydrates
but containing a substantial amount of protein and the
final fraction C showing strong peaks of protein and
carbohydrates. Fractions A, B and C so obtained were then
dialyzed against water for the de-salting purpose and then
freeze-dried to give the three powders G-A, G-B and G-C,
respectively.
These powders G-A, G-B and G-C were examined for
their antitumor activity against Sarcoma 180 solid tumor
by the bioassay method hereinbe~ore stated. The results
- 25 -
~3~
obtained are shown in Table 5 below.
Table 5
___ j _ .
Tes-t Dose Average Tumor Complete
Sample (mg/mouse tumor Inhibi- regression
x times) weight (g) tion
. . .. .
G-A 2 x 3 1.79 73.9 1/6
G-B 2 x 3 3.21 57.5 0/4
G-C 2 x 3 3.72 50.5 o/5
_ .. .__.--_ _ .
Control _ _ 7-55 ~ _ 0/6
It is observed that powder G-A showed the highest
antitumor potency among the other powders G-B and G-C
and could be recovered in the highest yield (usually of
about 2% based on the weight of the crude powder recovered
from the ion-exchange chromatography step), revealing
that the gel-filtration o~ crude powder of the active
substances with Sephadex G-75 column could afford the
highest efficiency in -the puri~ication of the acti~e sub-
stances.
To estimate approximately how much was the lower
limit of the molecular weights o~ the glycoprotein sub
stances present in the above-mentioned powder G-A, this
powder was subjected to gel-filtration with Sephadex G-50
(a product of Pharmacia Fine Chemicals Co.) with reference
to the molecular weights o~ tryptophane, vitamin B
- 26 -
~2~3~
cytochrome C and ovalbumin, when it was found that powder
G-A comprised glycoprotein substances having molecular
weights of not less than about 10,000. Thus, when a crude
powder of the active substances recovered from the preced-
ing stage of the anion-exchange chromatography is further
purified by gel-filtra-tion using a gel capable of fraction-
ating substances having molecular weights of 1500~100,000,
e.g. Sephadex G-75 to obtain the active glycoprotein sub-
stances having molecular weights of not less than 10,000,
it is found that said crude powder can be purified in an
industrial large scale, and be purified efficiently with-
out causing deactiva-tion of the active substances.
Powder G-A obtained above is further purified by
the following procedure.
One gram of powder G-A is dissolved in a small
amount of a 0~0.1 M phosphate buffer solution having a pH
of 700~7.5 and the resulting solution is passed through a
column packed with a gel for gel-filtration capable o~
fractionating substances having molecular weights of
10,000-2,000,000 which has been equilibrated wlth a
0~0.1 M phosphate buffer solution having a pH of 7.0-7.5,
e.g. Sephacryl S-400 (Sephacryl is a registered trade mark
of Pharmacia Fine Chemicals Co.) which is one obtained by
crosslinking allyldextran with N,N'-methylenebisacrylamide.
Elution is effected with a 0~0~1 M phosphate buffer solution
having a pH of 7.0~7,5 to yield fraction A corresponding
~13~
to molecular weights higher than 300,000~ lraction B
corresponding to molecular weights ranging 300,000~
150,000 and fraction C corresponding to molecular weights
lower than 150,000. Each o~ the three fractions A, B and
C is recovered in the form of dry powder S-A, S-B and
S-C, respectively, by subsequent dialysis and freeze-
drying~
The puri~ied glycoprotein powders (S-A), (S-B) and
(S-C) so obtained as in Example 4 were examined for their
antitumor activity against Sarcoma 180 solid tumor by the
bioassay method as stated hereinbefore. The test results
obtained are shown in Table 6 below~
Table 6
. .. _ ___ , .............. _ _ __ _
Test Dose Average Inhibi- Complete
Sample (mg/mouse wt. of tion regression
x times) tumors (g) ratio (%)
_ . , _ ~ .__ . .
S-~ 1 x 3 5.38 40.5 o/5
S-B 1 x 3 2.25 75~1 1/5
S-C 1 x 3 2.27 74.g 1/5
__ ~ _ _
Control ¦ - 9.05 _ 0/8
From the above table, it is seen that powder (S-A)
comprising the glycoprotein substances o~ higher than
300,000 in molecular weight showed a weaker antitumor
activity than that of powder (S-B) comprising the glyco-
protein substances of 150 9 000- 300,000 in molecular weight,
- 28 -
~2~3~
as well as of powder (S-C) comprising the glycoprotei-n
substances of 10,000~150,000 in molecular weight, and
also that powder (S-B) was not significantly different
from powder (S-C) in the degree of their antitumor activity.
We have made comparison in physical and chemical
properties of the antitumor agent consisting essentially
of water-soluble~ macromolecular glycoprotein substances
according to this invention as typified by those given
later in Example 5 with known similar antitumor agents
derived from shellfish which are disclosed in Japanese
Patent Publication No. 8088/82, Japanese Patent KOKAI
Mos. 41314/79 and 41315/79 and Journal of National Cancer
Institute, Vol. 60, No. 6, 1499-1500, June 1978 by
T. Sasaki et al. and found no similarity which makes it
possible to identify the antitumor agent of this invention
with those disclosed in literature. We believe therefore
that the antitumor agent according to this invention is
a new composition.
The antitumor agent according to this invention
may be used in combination with one or more other anti-
tumor agents, if desired. Particularly effective is a
combination with other antitumor agent which will enhance
the immunopotenciating effect. The antitumor agent
according to this invention has such particular advantages
that it has a broad antitumor spectrum without appreciable
cytotoxicity and that a noticeable tumor regression effect
- 29 -
8~i
can be achieved by adopting various administration routes
including intravenous, intraperitoneal, intracutaneous,
subcutaneous, and intratumoral administrations.
Characteristic ~eatures and advantages of the
process for the preparation of antitumor agent according
to this invention are summarily given below.
Firstly, the process of this invention is charac-
teri~ed in that it utilizes às starting material the
liquid portion which comes from the cooking of raw shell-
fish for the purpose of obtaining edible portions thereof
and which is usually discharged as waste, from which the
desired antitumor substances can be efficiently recovered.
This is based on our discovery that the desired antitumor
agent which consists essentially of water-soluble, macro-
molecular glycoprotein substances having a range of mole-
cular weights within the limits of from 10~000 to 300,000
can be recovered from shellfish through a high tempera-
ture treatment thereof without impairing the antitumor
activity of the desired antitumor agen-t and without affect-
ing edible portions of the shellfish. Such a discovery
is surprising in view of the prior art teaching that
extraction of antitumor substances from shellfish should
be carried out at a low temperaturep i.e. without hea-ting,
to avoid any deterioration of the desired substances.
Secondly, the process of this invention is much more
simple and inexpensive than the processes hitherto proposed
3~ -
~3~
as disclosed in the Japanese patent publication and KOKAI
publications above-referred to. In the prior art processes,
edible portions of raw shellfish must be used as starting
material and the extraction thereof at a low temperatare
requires such troublesome treatments as removal of liver
from edible portions of shellfish, cutting of the edible
portions into fine pieces, homogenization o~ the fine pieces
to serve efficient extraction, e.g. by sonication, and re-
moval of the consumed mass after extraction~ e.g. by centri-
fuging. A further disadvantage of the low-temperature
extraction of the prior art is that the resulting extract
contains not only the desired antitumor substances but
also many other substances, particularly macromolecular
organic substances, thus making the purification steps
required more complex. In contrast, the process of this
invention does not require any step for extraction and
subsidiaries thereto but may utilize the liquid portion
which comes from cooking of raw shellfish for recovery of
edible portions thereof and which is usually discarded as
waste. The liquid portion from the cooking step of raw
shellfish contains other substances than the desired anti-
tumor substances, particularly other organic macromolecular
substances in relatively low proportions in comparison with
the extract in the prior art processes and is therefore
purified more easily for the recovery and isolation of the
desired antitu~or substances therefrom.
8~-
The process of this invention is also advantageous
in view of environmental protection in that a substantial
amount of organic substances contained in the lia,uid
portion coming from cooking of raw shellfish can be re-
covered as useful product with the result that the organiccontents of the waste liquor are substantially reduced.
- 32 -
3~
~his invention is now illustrated with reference
to the following E~amples to which the invention is
limited in no way.
Example 1
One part (by weight) of raw scallop shellfish
(Patino~ecten 7essiensis) in the shells was charged into
a vessel continuously, into which 0.10 parts (by weight)
ol superheated steam at 105~110C was blown so that the
raw scallop shellfish was directly exposed to the blown
steam and steamed at 90~100C for 10 minutes. At the
bottom of the ~essel, there wa~ collected a volume of
the water condensate which was usually such one to be
discarded as waste liquor in the conventional cooking
process of scallops but which was now recovered according
to this invention as a first crop solution of the active
substances. The outer shells of raw scallop shellfish
used as the starting material had about 0.1 parts (by
weight) of the infesting acorn shells attached theretoO
The first crop solution of the active substances was
removed out of said vessel and then slowly cooled down
from 90C to 50~C and then i~mediately passed into a
spraying drier. This drier had an inlet for hot air
through which a stream of hot air at 280C was passed
into the drier, as well as an outlet for effluent gases
through which the effluent gases were discharged from
the drier at a temperature of 125~C or higher. The
- 33 -
first crop solution of the active substances which was
passed into the spraying drier was finely divided into
a spray within said drier and dried into powder in a
retention time of 45 seconds, and the resulting dried
powder was carried along with the effluent gases out of
the spraying drier and collected by a collector. In this
way, a first crude powder of the active substances was
obtained in a yield of 0.27~ by weight based on the whole
weight of the raw scallop shellfish employed.
Then, the shell-ligament portions were removed
from the shellfish by means of knife. The scallop liga-
ment so collected (50 kg) was placed into a volume (450 kg)
of a boiling solution containing 10% by weight of sodium
chloride in water and then boiled in the boiling saline
water for 20 minutes. With the same saline water, further
three 50 kg portions of the scallop ligament were treated
for the boiling process. This saline water wa~ recovered
as a second crop solution of the active substances. The
steamed meat portions and the boiled ligament portions
of the scallops were then frozen ~or sale and u~e. The
second crop solution of the active substances as recovered
wa~ slowly cooled down from 90C to 40C and processed in the
same manner as for the first crop solution of the active
substances by means of the spraying drier to ~ive a second
crude powder of the active substances in a yield of 0.20
by weight based on the whole weight of the scallop shell-
- 34 -
fish employed
Two parts by weight o~ the ~irst crude powder of
the active substances were admi~ed with one part by weight
o~ the ~econd crude powder of the active substances to
give a third crude powder of the active substances.
E~ample 2
The third crude powder (600 g) obtained in E~ample 1
was admixed with 3 kg of di~tilled water, and the resulting
aqueous suspension was centrifuged at 10,000 G for 20 minutes
at 5C to remove the insoluble parts there~rom~ The super-
natant solution ob-tained (3 litres) was passed for the de-
salting purpose into a column of 16 ~ of a gel-filtration
gel, Sephadex G-25 (a product of Pharmacia ~ine Chemicals
Co.) which had been swollen with water and then had been
equilibrated with a O.OlM phosphate buffer solution (pH 7.5)
containing 0.07 mol/litre of sodium chloride. This column
was subse~uently eluted with a further volume of the same
~.OlM phosphate buffer solution (p~ 7.5) containing
0.07 mol/litre o~ sodium chloride. The electroconductivity
of the eluate was measured continuously, and the fractions
which showed low electroconductivity were collected.
Such fractions containing the sodium chloride eluted out
showed a higher electric conductivity, whereas such
fractions containing the active substances but contain-
ing no sodium chloride ~howed a lo~ler electric conductivity.~he fractions of the lower electric conductivity were
35 -
combined together and passed through a column of 16~ of
an ion~exchanging gel, DEAE-Sepharose ~-6B which had been
equilibrated with a O.OlM phosphate buffer 301ution (pE 7.5)
containing 0.07 mol/litre of sodium chloride, for the
purpose of separating off the undesirable substances such
as polysaccharides and substances having low ion-affinity.
The effluent firstly running through the DEAE-~epharose
column wa~ discardedO After it was confirmed by means of
an ultra~violet ray monitor that the effluent did no longer
run out of the column~ the column having adsorbed the active
substances was then eluted with a volume of a O.OlM phos-
phate buffer solution (p~ 7.5) containing 0.25 mol/litre
of sodium chloride. The eluate fro~ the column was collected
and the fractions so obtained containing the active sub-
stances were combined together and dialyzed against de-
ionized water at 5C for two days for the de-salting
purpose.
The active fraction so dialyzed was then freeze-
dried to give 702 g of a fourth powder which was effective
against Sarcoma 180 in ICR mice9 with a dose of 400 mg/kg
intratumorally given once a day for 3 days. This powder
also showed a pronounced antitumor effect against Meth~A
in BAIB/c mice with a dose of 400 mg/kg intratumorally
given once a day for 6 days; it showed a 100~ inhibition
ratio with complete tumor regression in 6 of 6 mice
tested.
- 36 -
~L38~
xample 3
Eor the purpose of further purifying, the fourth
crude powder (30 g) obtained from repetition of the
procedure of Example 2 was dissolved in 150 m~ of a 0.
phosphate buffer solution (pH 7 5)~ and the resulting
solution was charged into a column (90 cm height x 14 cm
diameter) of a gel-filtration gel, Sephadex G75 which had
been equilibrated with another volume of the same O.lM
phosphate buffer solution (pH 7.5). The Sephadex G75
column having adsorbed the active substances was then
eluted with a further volume of the same O.IM phosphate
buffer solution (pH 7.5). The eluate was collected in
15 m~-fractions. The active fractions which were con-
taining the active substances having molecular weights
of not less than lOjO00 were combined together and dialyzed
against deionized water at 5C for 48 hours for the de-
salting. ~he active fraction so dialy~ed was then freeze-
dried to give 0.8 g of a fifth crude powder which showed
a strong antitumor activity (80.5~ inhibition ratio)
against Sarcoma 180 solid tumor with a dose o* 40 mg/kg
for 3 days.
Exam~le 4
For further purification and fractionation of the
fifth crude powder, the fifth crude powder (1.0 g) obtained
from repetition of the procedure of Example 3 was di~-
solved in 10 m~ of a O.OIM phosphate buffer solution
- ~7 -
lZ13~
(pH 7.5) and the resulting solution was chromatographed
in a column (50 cm height ~ 2,5 cm diameter) of a gel-
filtration gel, Sephacryl S-400 (a product of Pharmacia
~ine Chemicals Co.) which had been well equilibrated with
another volume of the same O.OlM phosphate buffer solution
(pH 7,5). The elution of this Sephacryl S-400 column was
effected with a further volume of the sa~e 0.OIM phosphate
buffer solution (pH 7O5) while the eluate was collected in
15 m~-fractions. The fractions NoO 28 to ~To. 40 were
combined together as the first glycoprotein frac-tion (A)
containing the acti~e substances of molecular weights of
higher than 300,000~ the fractions Nos. 41 to ~o. 65 were
combined together as the second glycoprotein fraction (B)
containing the active substances of molecula.r weights of
from 150,000 to 300~000; and the fra.ctions No. 66 to ~o. 95
were combined together as tke third glycoprotein fraction
(C) containing the active substances of molecular w~ights
of less than 150,000 but not less than 10,000. ~he
fractions (A), (B) and (C) were each dialyzed against
distilled water for 48 hours and then freeze-dried to
give 0.18 g o~ a purified glycoprotein powder (S-A)~
0.35 g of a purified glycoprotein powder (S-B) and 0.30 g
of a purified glycoprotein powder (S-C), respectively.
These glycopro-tein powders (S-A)~ (S-B) and (S-C)
were estimated for their antitumor activity against
Sarcoma 180 solid -tumor, when it was re~ealed that the
- 38 -
L3~313S
second glycoprotein powder (S-~) had a significant anti-
tumor activity against Sarcoma 180 solid tumor which was
substantially as high as that of the third glycoprotein
powder (S~C) and which was higher than the anti-
tumor potency of the fifth crude powder of the activesubstances as obtained in Example 3 t and also that the
first glycoprotein powder (S-A) sho~ed no significant
antitumor activity against Sarcoma 180 solid tumor.
~he mixed powder of the active glycoprotein powders
(~-3, 210 mg) and (S-C, 180 ~g) of Example 4 was dissolved
in a volume of distilled water~ followed by freeze-drying
to afford 0.39 g of a sample powder (S-D) of the active
glycoprotein. This sample powder (S-D) of the active
glycoprotein obtained according to this invention sho~ed
the following physico-chemical properties:-
(1) Elemental analy~is: C 43.2~, H 6 . 5~o~ ~ 10~ l~o, P 0.12
S 0,l~o,ash l~o by weight.
(2) Molecular weight: About lO~OOG to about 300,000 as
measured by gel-filtration chro-
matography.
(3) Melting point: No definite melting point with
decomposing at 235C.
(4) ~pecific optical [a]20 - 15~7 (c=0.5~o b wt.
rotation: in water~
- 39-
~381~
(5) Ultraviolet absorption A solution of 0.4 mg of the
~pectrum:
sample powder per m~ of water
gave the W absorption spectrum
as shown in Figure 4, with a
characteristic absorption peak
H20
~ max at 278 nm
(6) Infrared absorption As shown in ~igure 5, with
spectrum (pelleted
in ERr): characteristic absorption peaks
at 3500~~300, 1650, 1540 and
1400 cm 1.
(7) Solubility: ~oluble easily in water but
insoluble in organic solvents
such as methanol, ethanol and
acetone.
~8) Color reactions: Positive in biuret reaction,
~anthoproteic reaction, phenolic
reagent reaction, anthrone-
~ul~uric acid reaction a~d
phenol-sulfuric acid reaction;
but pseudo-positive in cysteine-
sul~uric acid reaction.
(9) Acidic or basic nature: Amphoteric electrolyte.
~10) Substance color and White-colored solid with or
appearance:
without faint brown tinge.
- 40 ~
~2~3135
(11) Amino acids in When hydrolyzed in 6N HC~ at
hydrolysate:
105~110C for 24 hours, the re-
sulting hydrolysate contains at
least the following amino acids:-
aspartic acid, hydroproline,
threonine, ssrine, glutamic acid,
proline, glycine 9 alanine, cysteine,
valine9 methionine9 isoleucine,
leucine, tyrosine 7 phenylalanine,
lysine, arginine, histidine,
hydro~ylysineO
(12) Carbohydrates in When hydrolyzed in 2~ HC~ at
hydrolysate:
80~90C for 10 hours, ~ollowed
by the removal of the amino acids
from the hydrolysate by ion-
exchange chromatography and
further by hydrogenation of the
amino acids-free hydrolysate,
the hydrogenated hydrolysate con-
tains at least the following carbo-
hydrate~:
fructoseg mannose, fucose,
inositol, galactose.
(13) Protein content: The protein content amounts to
60.5~ by weight in terms of fatal
bovine serum albumin when measured
- 41 -
~%~L388~
by Lowry-~olin method ~lith
coloration by phenolic reagent.
(14) Carbohydrate ~ontent: ~he carbohydrate content amounts
to l9.g~ by weight in terms of
glucose when mea~ured by the
phenol-sulfuric acid method.
(15) Nucleic acid content: ~he nucleic acid content amounts
to 0.8~ or less by weight in
terms of adenylic acid when
measured according to the di-
phenylamine method by Dische
reactionO
Further, to evaluate the biological properties of
sample powder (S-D) of this in~ention, the antitumor effect
f sample po~lder (S-D) on Sarcoma 180 solid tumor was
determined by the bioassay method hereinbefore stated9
by injecting an aqueous solution of said sample powder
directly into the tumor site at various dosages three times,
namely on days of 5, 7 and 9 a~ter the tumor inoculation
in ICR mice, The test results obtained are tabulated in
the following table~
- ~2 -
~Z~3~
Tabl~ 7
Dosage Average
Test (mg/mouse wt.of Inhibition Complete
SamPle x times) _m ~ ratio (~ re~ression
S-D 2 x 3 1.97 73.9 1/6
S-D 1 x 3 1.61 78.1 1/5
3-D0.5 ~ 3 0 05 99 3 3/4
S-D0~2 x 3 3~40 66.9 1/3
S-D0.1 x 3 4 55 38.] 0/5
_. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Control(untreated) 7.55 0 0/8
. .
Administration by various routes such as intravenous,
intraperitoneal and subcutaneous injections of sample powder
(S-D) at various dosages (0 5~5 mg/mouse x 1~3 times) in
Sarcoma 180 solid tumor-bearing ICR mice also e~hibited a
signi~icant antitumor activity (50%-91~ in inhibition ratios).
~urthermore~ to examine any direct cytocidal effect
of the water-soluble glycoprotein substances of this inven
tion on tumor cell, mouse ~ymphoma ~ 5178 Y cells were
cultured at the concentration of 5 x 105 cells/m~ in RPMI
medium 1640 containing glycoprotein powder (S-B) or (S-C)
of Example 4 or sample powder (S-D) of this Egample 5 at
concen-trations of 40 to 200 mg/mQ. After culturing at 37C
for 48 hours, the viability of the tumor cells was egamined
by trypan blue staining9 when it was observed that the tumor
cells showed 100~ viability, revealing that glycoprotein
43 -
~3~
powders (S-~), (S-C) and (S-D) had no direct cytocidal
effect on the tumor cell.
xample 6
This E~ample illustrates the antitumor activity of
the water-soluble glycoprotein substances of this invention
on the other tumors than Sarcoma 180 solid tumor.
The tumor cells (4 x 106 cells/mouse) of Ehrlich
carcinoma, ~eukemia SN 369 ~T~ reticulum cell sarcoma or
Meth/A Fibrosarcoma were subcutaneously injected into the
inguinal part of ICR mice or ~AIB/c mice (6 weeks aged).
One week after the tumor cell inoculation, the tumor cells
had grown to solid tumors. A solution in physiological
saline of sample powder (S D) obtained in Example 5 was
injected into the tumor site at a u~it dosage of 5mg/mouse
three times 9 namely on days 5, 7 and 9 after the tumor
cell inoculation. ~ive weeks a-fter the tumor cell
lnoculation, the mice were sacrificed and the tumors
dissected out and weighed~ The inhibition ratio (~ was
calculated as compared with the control groups of mice
which received the tumor cell inoculation and injection
of physiological saline water containing no test sample.
The test results obtained are summari~ed in Table
8 below.
- 44 -
~138~
Table 8
Inhibition ratio (~)
NT~ reticu-
Test Ehrlich ~eukemia lum cell Meth/A
substance carcinoma S~-36 sarcoma ~ibrosarcoma
(Mice tested) (ICR mice) (ICR mice) (ICR mice) (BALB/c mice~
S-D 80.3 95-5 83.5 90.2
_
Control 0
. . . ~
Exam~le 7
,_ _
Raw horned turban shellfish ( atillus cornutus) in
their shells was steamed and boiled in -the same manner as
in Example 1. The water condensate collected was spray-
dried in the same manner as in Egample 1. The resulting
dried powder was processed with Sephadex G-25 and with
DEAE-Sepharose CI~6~ in the same manner as in Example 2,
then with Sephade~ G75 in the same manner as in Egample 3
and with Sephacryl ~-400 in the same manner as in Example 4
and then blended in the same manner as in E~ample 5 to give
a ~lhite-colored powder o~ water-soluble glycoproteins having
molecular weights of from 10~000 to 300,000 as measured by
the gel-filtration chromatography. This purified glyco-
protein powder showed a significant antitumor activi-ty as
it gave 65~ inhibition ratio against Sarcoma 180 solid
tumor when tested by the bioassay method stated herein-
before at a dosage of 400 mg/kg ~ 3 times.
- 45 -
38~
Example 8
Raw abalone shellfish (Haliotis discus) in their
shells was steamed ana boiled in the same manner as
in Example 1. The water condensate collected was
spray-dried in the same manner as in Example 1. The
resulting dried powder was processed with Sephadex G-25
and with DEAE-Sepharose CI-6B in the same man~ner as in
Example 2, then with Sephadex G75 in the same manner as
in Example 3 and with Sephacryl S-400 in the same manner
as in Example 4, and then blended in the same manner as
in E~ample 5 to give a white-colored powder of water-
soluble glycoproteins having molecular weights of from
10,000 to 300,000 as measured by -the gel-filtration
chromatography. This purified glycoprotein powder showed
a significant antitumor activity as it gave 7~ inhibition
ratio against Sarcoma 180 solid tumor when tested by the
bioassay method stated hereinbe~ore at a dosage o~ 400 mg/kg
x 3 times.
Raw Asian hard clam shellfish (Meretrix lusoria) in
their shells was s-teamed and boiled in the same manner as
in Example 1. The water condensate collected was spray-
dried in the same manner as in Example 1~ The resulting
dried powder was processed with Sephadex G-25 and with
DEAE-Sepharose CL-6~ in the same manner as in Example 2,
then with Sephadex G75 in the same manner as in Example 3
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~2~3i~8S
and with Sephacryl S~400 in the same manner as in E~ample 4,
and then blended in the same manner as in Example 5 to
give a white~colored powder of water~soluble glycoproteins
having molecular weights of from 10,000 to 300~000 as
measured by the gel-filtration chromatography. This
purified glycoprotein powder showed a significant anti-
tumor activity as it gave 67~ inhibition ratio against
Sarcoma 180 solid tumor when tested by the bioassay method
stated here.inbefore at a dosage of 400 mg/kg ~ 3 times.
Example 10
The prccedure~ of Examples 2, 3, 4 and 5 were
repeated using the first crude powder of the active sub-
stances obtained in Example 1 (by spray-drying the first
crop solution of the active substances, namely the water-
condensate recovered from the steaming of scallops) inplace of the third crude po~der of the active substances
of Example 1, There was afforded a white~colored powder
of water-soluble glycoproteins which e~hibited the physico-
chemical properties substantially the same 2S those of
s~mple powder (S-D) of E~ample 5 and also gave an anti-
tumor activity against Sarcoma 180 solid tumor as high as
that of ~ample powder (S-D~ of Example 5.
The procedures of Examples 2, 3, 4 and 5 were
repeated using the second crude powder of the active
substances obtained in Example 1 (by spray-drying the
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~38~3~
second crop solution of the active substances as recovered
from the boiling of the scallop ligaments with saline
water) in place of the third crude powder of the active
substances OI Example lo ~here was afforded a white-
colored powder of water-soluble glycoproteins which
e~hibited the physico-chemical properties substantially
the same as those of sample powder (S~D) of Example 5
and also gave an antitumor activity against Sarcoma 180
solid tumor as high as that of sample powder (S-D) of
Egample 5.
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